基于核因子-κB受体活化因子受体/骨保护素探讨复方雷公藤外敷剂改善Ⅱ型胶原诱导型关节炎模型大鼠骨破坏

Study on Compound Tripterygium Wilfordii External Application on the Improvement of Bone Destruction in Rats with Collagen-Induced Arthritis Based on RANKL/OPG

目的:观察复方雷公藤外敷剂对Ⅱ型胶原诱导型关节炎模型大鼠的骨形态学影响,探讨其可能的作用机制。方法:将50只雌性SD大鼠随机分为正常组(NOR组)、模型组(CIA组)、复方雷公藤外敷剂组(TWE组)、雷公藤多苷组(TWG组)、扶他林乳胶剂组(VOT组),每组10只。除NOR组外,对其余大鼠进行尾根部皮下注射Ⅱ型胶原诱导,7 d后加强免疫造模。TWE组以0.60 g/(kg·d)外用、TWG组6.25 mg/(kg·d)灌胃、VOT组乳胶剂0.016 mL/(kg·d)外用,NOR组与CIA组以生理盐水涂抹膝踝关节,连续给药28 d。观察大鼠一般状态、双下肢肿胀程度;观察各组膝、踝关节病理形态,借助微型计算机断层扫描(micro-CT)观察骨质改变,采用酶联免疫吸附法(ELISA)测定、蛋白免疫印迹法(Western Blot)观察外周血及组织骨破坏与骨保护相关因子与蛋白表达。结果:与NOR组比较,CIA组一般状态不佳,关节炎评分与足趾容积均显著增高,micro-CT显示踝关节、跖趾关节破坏严重,血清MMP1、MMP13、RANKL显著增高,滑膜组织中RANKL/OPG显著升高。与CIA组相比,各治疗组随着给药时间的增加,关节炎评分与足趾容积均有下降,micro-CT显示踝关节、跖趾关节骨表面积/骨体积比值低于模型组,差异有统计学意义(P<0.05),其中TWE组与VOT组改善最为明显,差异有统计学意义(P<0.01);各治疗组血清MMP1、MMP13、RANKL、关节组织中RANKL/OPG均低于CIA组。结论:复方雷公藤外敷剂能够改善模型大鼠关节骨破坏,其作用机制可能与降低MMP1、MMP13、RANKL水平,抑制RANKL/OPG,调控骨平衡有关。

Objective:To observe the efficacy of compound tripterygium wilfordii external application on bone morphology of rats with typeⅡcollagen-induced arthritis and to explore its possible mechanism.Methods:50 female SD rats were randomly divided into normal group(NOR group),model group(CIA group),compound tripterygium wilfordii external application group(TWE group),tripterygium wilfordii glycosides group(TWG group)and voltaren cream group(VOT group)with 10 rats in each group.Except for the NOR group,rats in the other groups were induced by subcutaneous injection of typeⅡcollagen into the tail root,and strengthened immune after 7 d.The rats in the TWE group(0.60 g/(kg·d)),TWG group(6.25 mg/(kg·d))and VOT group(latex 0.016 mL/(kg·d))were treated for 28 d,while the CON group and CIA group coated knee and ankle joint with normal saline for 28 d.The general state and the swelling degree of both lower limbs of rats were observed,the bone changes were observed by microcomputer tomography(micro-CT),and the expression of bone protection related factors and proteins in peripheral blood and tissue were observed by enzyme linked immunosorbent assay(ELISA)and Western Blot.Results:Compared with NOR group,the general condition of CIA group was poor,the arthritis index and toe volume were significantly increased,micro-CT showed that ankle joint and metatarsophalangeal joint were seriously damaged,the MMP1,MMP13,RANKL in serum and the RANKL/OPG in synovium were significantly increased.Compared with the CIA group,the arthritis index and toe volume of each treatment group decreased with the increase of administration time,and micro-CT showed that the bone surface area/bone volume ratio of ankle joint and metatarsophalangeal joint was lower than that of the CIA group(P<0.05),especially in TWE group and VOT group is obvious(P<0.05).The serum MMP1,MMP13,RANKL and RANKL/OPG in joint tissue in each treatment group were lower than those in CIA group.Conclusion:Compound tripterygium wilfordii external application can effectively improve the destruction of articular bone in CIA rats,and its mechanism may be related to reducing the levels of MMP1,MMP13 and RANKL,inhibiting RANKL/OPG and regulating bone balance.